Methods of treating drug resistant and other tumors by administering 6,7-dialkoxy quinazoline derivatives

ABSTRACT

Methods employing and uses of a compound of formula (I) in inhibiting the growth of a tumor cell in a subject in need thereof. Methods employing and uses of a compound of formula (I) in treating pancreatic cancer in a subject in need of treatment for pancreatic cancer. Methods employing and uses of a compound of formula (I) in treating HER-2 positive breast cancer in a subject in need of treatment for HER-2 positive breast cancer. Methods employing and uses of a compound of formula (I) in treating drug resistant non-small cell lung cancer in a subject in need of treatment for drug resistant non-small cell lung cancer. Each of these methods can include administering to the subject an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. 
     Method of manufacturing a medicament including a compound of formula (I) for treating a subject suffering from growth of a tumor cell; for treating a subject suffering from pancreatic cancer; for treating a subject suffering from HER-2 positive breast cancer; or for treating a subject suffering from drug resistant non-small cell lung cancer.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of Ser. No. 61/225,419, filed Jul.14, 2009 in the United States and which application is incorporatedherein by reference.

FIELD OF THE INVENTION

Methods employing and uses of a compound of formula (I) in inhibitingthe growth of a tumor cell in a subject in need thereof. Methodsemploying and uses of a compound of formula (I) in treating pancreaticcancer in a subject in need of treatment for pancreatic cancer. Methodsemploying and uses of a compound of formula (I) in treating HER-2positive breast cancer in a subject in need of treatment for HER-2positive breast cancer. Methods employing and uses of a compound offormula (I) in treating drug resistant non-small cell lung cancer in asubject in need of treatment for drug resistant non-small cell lungcancer. Each of these methods can include administering to the subjectan effective amount of a compound of formula (I) or a pharmaceuticallyacceptable salt thereof.

Method of manufacturing a medicament including a compound of formula (I)for treating a subject suffering from growth of a tumor cell; fortreating a subject suffering from pancreatic cancer; for treating asubject suffering from HER-2 positive breast cancer; or for treating asubject suffering from drug resistant non-small cell lung cancer.

BACKGROUND OF THE INVENTION

Most of the treatment regimes of the past for cell proliferationdiseases such as psoriasis and cancer utilize compounds which inhibitDNA synthesis. Such compounds are toxic to cells and their beneficialeffects can be derived when they show selectivity to tumor cells. Inrecent years it has been discovered that a cell may become cancerous byvirtue of the transformation of a portion of its DNA into an oncogene,i.e., a gene which, on activation, leads to the formation of malignanttumour cells (Bradshaw, Mutagenesis, 1986, 1:91). Several oncogenesencode tyrosine kinase enzymes and certain growth factor receptors arealso tyrosine kinase enzymes (Larsen et al., Ann. Reports in Med. Chem.1989, Chapt. 13).

Receptor tyrosine kinases are important in the transmission ofbiochemical signals which initiate cell replication. They possess anextra cellular binding domain for growth factors such as an epidermalgrowth factor and an intracellular portion which functions as a kinaseto phosphorylate tyrosine amino acids in proteins and hence to influencecell proliferation. Members of the ErbB family of receptor tyrosinekinases are mediators of cell growth, differentiation and survival thathave been implicated in cancer. The receptors are over expressed incertain tumor cells. For example, it is known that such kinases arefrequently present in common human cancers such as breast cancer(Sainsbury et al., Brit, J. Cancer, 1988, 58:458) and gastro intestinalcancers such colon, rectal and stomach cancers (Bolen et al., OncogeneRes., 1987, 1:149). It was discovered that Tyrosine Kinase activity (TKactivity) is more frequently detectable in malignant cells than innormal cells (Hunter, Cell, 1987, 50:823).

More recently, it has been shown that Epidermal Growth Factor Receptor(EGFR) which possesses TK activity is over expressed in many humancancers such as brain, lung squamous cell, bladder, gastric, breast,head & neck, oesophageal, thyroid and the like. (W. J. Gullick, Brit.Med. Bull. 1991, 47:87). The receptor family includes four distinctmembers, including epidermal growth factor receptor (EGFR or ErbB1),HER2 (ErbB2 or p185^(neu)), HER3 (ErbB3) and HER4 (ErbB4 or tyro2). TheHER (ErbB) family belongs to the subclass I receptor tyrosine kinasesuperfamily and consists of three distinct receptors, HER2, HER3, andHER4. Sequences of these receptors can be found in U.S. Pat. No.5,183,884 (erbB3/HER3); U.S. Pat. No. 5,811,098 (HER4/Erb4 receptor);erbB2/HER2: Semba et al. (1985) Proc. Natl. Acad. Sci. USA 82:6497-6501(designating the gene c-erbB-2); Coussens et al. (1985) Science230:1132-1139 (designating the gene HER2); or King et al. (1985) Science229:974-976.

Another receptor tyrosine kinase that is associated with cancer is theVEGF (vascular endothelial growth factor) receptor, the sequence ofwhich is disclosed in U.S. Pat. No. 5,332,671.

A strategy to inhibit EGFR-TK activity has been exploiting smallsynthetic molecules (Arteaga C L, Exp. Cell Res., 2003, 284:122-130).Certain quinazoline derivatives like gefitinib (IRESSA®, Astra Zeneca),erlotinib (OSI-774, TARCEVA®), PD-183805, PKI-166, EKB-569, PD-168393,CGP-59362 have been have been investigated for possible treatmentoptions for several forms of cancer (Baselga et al., Oncology 2002, 63:6-16, Cohen R B., Clin. Colorectal Cancer, 2003, 2:246-251). TheEuropean patent applications namely EP 0566226, EP0602851A1, EP 0635507A1, EP 0635498 A1, EO 0520722 A1 disclose certain quinazolinederivatives possessing anti-cancer properties and that inhibit TK.

U.S. Pat. Nos. 5,475,001, 5,457,105, 5,616,582, 5,770,599, 5,747,498,and 6,900,221 disclose quinazoline derivatives with structural featuressuch as a substituted anilino moiety in the 4-position and a variety offunctionalized alkyl groups in the 6- and 7-positions of the quinazolinenucleus. Specifically U.S. Pat Nos. 5,457,105, 5,616,582 discloseN-(3-Chloro-4-fluorophenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(Gefitinib) and U.S. Pat. Nos. 5,747,498 and 6,900,221 discloseN-(3-Ethylnylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine(Erlotinib). WO 2005/070909, WO 2007/060691 A₂ and WO 06/090413 disclosevariations in synthesis or polymorphic forms of these two popularanti-cancer drugs.

Nonetheless, there remains a need for additional cancer therapies.

SUMMARY OF THE INVENTION

The present invention includes a method of treating drug resistantnon-small cell lung cancer in a subject in need of treatment for drugresistant non-small cell lung cancer. The method includes administeringto the subject a compound of formula (I):

in which:

-   R¹ is:

-   R² is —CH₃ or —CH₂CH₃; or a pharmaceutically acceptable salt of the    compound of formula (I). Suitable pharmaceutically acceptable salts    include a monohydrochloride, a dihydrochloride, or a mixture    thereof. This method can employ a compound of formula (I) in which    R¹ is:

and R² is —CH₃. This method can employ a compound of formula (IA):

The present invention includes the use of a compound of formula I or apharmaceutically acceptable salt thereof for treating a subjectsuffering from drug resistant non-small cell lung cancer. The presentinvention also includes a use of a compound of formula I or apharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from drug resistantnon-small cell lung cancer.

The present invention includes a method of treating HER2 positive breastcancer in a subject in need of treatment for HER2 positive breastcancer. The method includes administering to the subject a compound offormula (I), which compound is described above, or a pharmaceuticallyacceptable salt thereof. The present invention includes the use of acompound of formula I or a pharmaceutically acceptable salt thereof fortreating a subject suffering from HER2 positive breast cancer. Thepresent invention also includes a use of a compound of formula I or apharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from HER2 positive breastcancer.

The present invention includes a method of treating pancreatic cancer ina subject in need of treatment for HER2 positive breast cancer. Themethod includes administering to the subject a compound of formula (I),which compound is described above, or a pharmaceutically acceptable saltthereof. The present invention includes the use of a compound of formulaI or a pharmaceutically acceptable salt thereof for treating a subjectsuffering from pancreatic cancer. The present invention also includes ause of a compound of formula I or a pharmaceutically acceptable saltthereof for the manufacture of a medicament for treating a subjectsuffering from pancreatic cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph illustrating the results of experiments that comparedthe compound of formula IA to erlotinib and gemcitabine in causing tumorregression.

FIG. 2 shows results of immunohistochemical analysis of pancreaticsections (anti-angiogenic activity) for animals treated with thecompound of formula IA, erlotinib and gemcitabine in causing tumorregression.

FIG. 3 is a graph illustrating the results of experiments that comparedthe compound of formula IA to lapatinib in causing tumor regression.

FIG. 4 is a graph illustrating the results of experiments thatestablished regression of drug resistant tumors caused by the compoundof formula IA.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

As used herein, the term “therapeutically effective dose” refers to adose of the present compound of formula I (e.g., formula IA) thatprovides an amount of present compound in and around a tumor celleffective to achieve a desired biological activity of the presentcompound, e.g., reducing the rate of or stopping the growth of the tumorcell or killing the tumor cell. Desired biological activities includeinhibiting EGFR tyrosine kinase of the tumor cell. Inhibiting the growthof a cell can include, for example, reducing the number of divisions thecell undergoes (e.g., to zero), killing the cell, reducing thepopulation of a plurality of such cells, or the like.

As used herein, the term “effective amount” of the present compound offormula I (e.g., formula IA) is an amount sufficient to prevent, treat,reduce and/or ameliorate the symptoms and/or underlying causes of canceror growth of a tumor cell. In some instances, an “effective amount” issufficient to eliminate the symptoms of cancer or the growth of thetumor cell and, perhaps, overcome the cancer (e.g., put the cancer intoremission) or the growth of the tumor cell. In the context of thepresent invention, the terms “treat” and “therapy” and the like refer toalleviate, slow the progression, prophylaxis, attenuation or cure ofexisting disease (e.g., cancer or growth of a tumor cell). Prevent, asused herein, refers to putting off, delaying, slowing, inhibiting, orotherwise stopping, reducing or ameliorating the onset of such diseasesor disorders (e.g., cancer or growth of a tumor cell). It is preferredthat a large enough quantity of the agent be dosed in non-toxic levelsin order to provide an effective level of activity within the subjectagainst the disease. The method of the present invention may be usedwith any mammal. Suitable mammals include, but are not limited to rats,cats, dogs, horses, cows, sheep, pigs, and, preferably, humans.

The Present Methods and Uses

The present invention includes a variety of methods employing thecompound of the present invention. In an embodiment, the present methodincludes a method of inhibiting the growth of a tumor cell in a subjectin need thereof. The tumor cell can be a pancreatic cancer cell, a HER2positive breast cancer cell, or a drug (e.g., erlotinib or gefitinib)resistant non-small cell lung cancer cell. The present inventionincludes the use of a compound of formula I or a pharmaceuticallyacceptable salt thereof for treating a subject suffering from growth ofa tumor cell, such as pancreatic cancer cell, a HER2 positive breastcancer cell, or a drug (e.g., erlotinib or gefitinib) resistantnon-small cell lung cancer cell. These embodiments can includeadministering to the subject an effective amount of a compound offormula (I), which compound is described above, or a pharmaceuticallyacceptable salt thereof. These embodiments can employ a compound offormula (I) in which R¹ and R², are as described in embodiments above.These embodiments can employ a compound of formula (IA), which compoundis described above, or a pharmaceutically acceptable salt thereof. Theseembodiments can employ a pharmaceutical composition including a compoundof formula (I) or a pharmaceutically acceptable salt thereof. In anembodiment, the tumor cell is a pancreatic cancer cell. In anembodiment, the tumor cell is a HER2 positive breast cancer cell. In anembodiment, the tumor cell is a drug resistant non-small cell lungcancer cell. In an embodiment, the drug is erlotinib, gefitinib, or aplurality thereof.

The present invention also includes a use of a compound of formula I ora pharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from growth of a tumor cell,such as pancreatic cancer cell, a HER2 positive breast cancer cell, or adrug (e.g., erlotinib or gefitinib) resistant non-small cell lung cancercell. This embodiment can employ a compound of formula (I) in which R¹and R², are as described in embodiments above. This embodiment canemploy a compound of formula (IA), which compound is described above, ora pharmaceutically acceptable salt thereof.

In an embodiment, the present method includes a method of treating drugresistant non-small cell lung cancer in a subject in need of treatmentfor drug resistant non-small cell lung cancer. The present method alsoincludes the use of a compound of formula I for treating a subjectsuffering from drug resistant non-small cell lung cancer. In anembodiment, the drug is erlotinib, gefitinib, or a plurality thereof.These embodiments of the method can include administering to the subjectan effective amount of a compound of formula (I), which compound isdescribed above, or a pharmaceutically acceptable salt thereof. Theseembodiments of the method can employ a compound of formula (I) in whichR¹ and R², are as described in embodiments above. These embodiments ofthe method can employ a compound of formula (IA), which compound isdescribed above, or a pharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I ora pharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from drug (e.g., erlotinibor gefitinib) resistant non-small cell lung cancer. This embodiment canemploy a compound of formula (I) in which R¹ and R², are as described inembodiments above. This embodiment can employ a compound of formula(IA), which compound is described above, or a pharmaceuticallyacceptable salt thereof.

In an embodiment, the present method includes a method of treating HER2positive breast cancer in a subject in need of treatment for HER2positive breast cancer. The present method also includes the use of acompound of formula I for treating a subject suffering from HER2positive breast cancer. These embodiments of the method can includeadministering to the subject an effective amount of a compound offormula (I), which compound is described above, or a pharmaceuticallyacceptable salt thereof. These embodiments of the method can employ acompound of formula (I) in which R¹ and R², are as described inembodiments above. These embodiments of the method can employ a compoundof formula (IA), which compound is described above, or apharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I ora pharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from HER2 positive breastcancer. This embodiment can employ a compound of formula (I) in which R¹and R², are as described in embodiments above. This embodiment canemploy a compound of formula (IA), which compound is described above, ora pharmaceutically acceptable salt thereof.

In an embodiment, the present method includes a method of treatingpancreatic cancer in a subject in need of treatment for pancreaticcancer. The present method also includes the use of a compound offormula I for treating a subject suffering from pancreatic cancer. Theseembodiments of the method can include administering to the subject aneffective amount of a compound of formula (I), which compound isdescribed above, or a pharmaceutically acceptable salt thereof. Theseembodiments of the method can employ a compound of formula (I) in whichR¹ and R², are as described in embodiments above. These embodiments ofthe method can employ a compound of formula (IA), which compound isdescribed above, or a pharmaceutically acceptable salt thereof.

The present invention also includes a use of a compound of formula I ora pharmaceutically acceptable salt thereof for the manufacture of amedicament for treating a subject suffering from pancreatic cancer. Thisembodiment can employ a compound of formula (I) in which R¹ and R², areas described in embodiments above. This embodiment can employ a compoundof formula (IA), which compound is described above, or apharmaceutically acceptable salt thereof.

Unexpected Advantages of the Compounds of the Present Invention

Compounds of the present invention exhibit unexpected advantages inmethods of the present invention. For example, compounds of the presentinvention (e.g., a compound of formula (IA)) has exhibited unexpectedlysuperior anti-cancer and anti-proliferative effects compared to knowncompounds such as erlotinib, gefitinib, and lapatinib.

The compounds of the present invention (e.g., a compound of formula(IA)) exhibited activity against numerous kinases in in vitro assays.The compound of formula (IA) was evaluated for inhibition of 80 kinasesin an in vitro kinase profiling study and significantly inhibited 15 ofthem.

The compound of formula (IA) was evaluated in an in vivo model ofpancreatic cancer and found to be unexpectedly superior to erlotinibalone or in combination with gemcitabine. The compound of formula (IA)caused regression of subcutaneous pancreatic tumors in nude mice. Thetumor regression observed was superior to that caused by the existingdrugs erlotinib and gemcitabine.

The compound of formula (IA) was evaluated in an in vivo model of HER2positive breast cancer. The tumor regression observed with the compoundof formula (IA) in SCID mouse xenograft models of HER2 positive breastcancer was superior to that of Lapatinib, an approved drug of choice fortreatment of HER2 positive breast cancer.

The compound of formula (IA) was evaluated in an in vivo model of drug(e.g., erlotinib or Gefitinib) resistant non-small cell lung cancer. Thecompound of formula (IA) showed significant anticancer activity inanimals implanted with erlotinib/gefitinib resistant H1975 cells. Tumorregression was observed after withdrawal of treatment with the compoundof formula (IA).

Embodiments of the Present Methods

In an embodiment, a compound of the present invention can be employed toinhibit the growth of a cell including a tyrosine kinase. For example, acompound of the present invention can inhibit the growth of a tumor cellor malignant cell (e.g., a pancreatic cancer cell, a HER2 positivebreast cancer cell, or a drug (e.g., erlotinib or gefitinib) resistantnon-small cell lung cancer cell) that expresses a tyrosine kinase, suchas an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, ora VEGF receptor tyrosine kinase. Such a tumor cell or malignant cell canover express the receptor tyrosine kinase. In an embodiment, the cell isa drug (e.g., erlotinib or gefitinib) resistant non-small cell lungcancer cell. In an embodiment, the cancer cell is a pancreatic cancercell. In an embodiment, the cancer cell is a breast cancer cell thatexpresses HER2. In certain embodiments, the present compound can inhibitthe growth of the tumor cell or malignant cell in vitro, ex vivo, or invivo.

In an embodiment, the method can include contacting a cell expressingthe tyrosine kinase with a compound of the present invention. In anembodiment, the method can include contacting a tumor containing a cellexpressing the tyrosine kinase with a compound of the present invention.In certain embodiments, the present method can include contacting invitro, ex vivo, or in vivo.

In an embodiment, the method can include contacting a tumor cell ormalignant cell (e.g., a pancreatic cancer cell, a HER2 positive breastcancer cell, or a drug (e.g., erlotinib or gefitinib) resistantnon-small cell lung cancer cell) that expresses a tyrosine kinase, suchas an epidermal growth factor (EGF) receptor tyrosine kinase, an Erb-2tyrosine kinase, an Erb-3 tyrosine kinase, an Erb-4 tyrosine kinase, ora VEGF receptor tyrosine kinase with a compound of the presentinvention. In certain embodiments, the method can include contacting apancreatic cancer cell, a HER2 positive breast cancer cell, or a drugresistant (e.g., erlotinib or gefitinib resistant) non-small cell lungcancer cell with a compound of the present invention. In an embodiment,the method can include contacting a drug resistant (e.g., erlotinib orgefitinib resistant) non-small cell lung cancer cell with a compound ofthe present invention. In an embodiment, the method can includecontacting a pancreatic cancer cell with a compound of the presentinvention. In an embodiment, the method can include contacting a breastcancer cell that expresses HER2 with a compound of the presentinvention. In certain embodiments, the present method can includecontacting in vitro, ex vivo, or in vivo.

In an embodiment, the method can include contacting a tumor (e.g., apancreatic cancer tumor, a HER2 positive breast cancer tumor, or a drug(e.g., erlotinib or gefitinib) resistant non-small cell lung cancertumor) that expresses a tyrosine kinase, such as an epidermal growthfactor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, anErb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptortyrosine kinase with a compound of the present invention. In certainembodiments, the method can include contacting a pancreatic cancertumor, or a HER2 positive breast cancer tumor, or a drug (e.g.,erlotinib or gefitinib) resistant non-small cell lung cancer tumor witha compound of the present invention. In an embodiment, the method caninclude contacting a drug resistant (e.g., erlotinib or gefitinibresistant) non-small cell lung cancer tumor with a compound of thepresent invention. In an embodiment, the method can include contacting apancreatic cancer tumor with a compound of the present invention. In anembodiment, the method can include contacting a breast cancer tumor thatexpresses HER2 with a compound of the present invention. In certainembodiments, the present method can include contacting in vitro, exvivo, or in vivo.

In an embodiment, the method can include administering the compound ofthe present invention to a subject (e.g., a mammal or a warm bloodedanimal) having a tumor containing a cell (e.g., a pancreatic cancercell, a HER2 positive breast cancer cell, or a drug (e.g., erlotinib orgefitinib) resistant non-small cell lung cancer cell) expressing thetyrosine kinase. In an embodiment, the method can include administeringthe compound of the present invention to a subject (e.g., a mammal or awarm blooded animal) having a tumor cell or malignant cell (e.g., apancreatic cancer cell, a HER2 positive breast cancer cell, or a drug(e.g., erlotinib or gefitinib) resistant non-small cell lung cancercell) that expresses a tyrosine kinase, such as an epidermal growthfactor (EGF) receptor tyrosine kinase, an Erb-2 tyrosine kinase, anErb-3 tyrosine kinase, an Erb-4 tyrosine kinase, or a VEGF receptortyrosine kinase. In certain embodiments, the method can includeadministering the compound of the present invention to a subject (e.g.,a mammal or a warm blooded animal) having pancreatic cancer, HER2positive breast cancer, or drug resistant (e.g., erlotinib or gefitinibresistant) non-small cell lung cancer. In an embodiment, the method caninclude administering the compound of the present invention to a subject(e.g., a mammal or a warm blooded animal) having drug resistant (e.g.,erlotinib or gefitinib resistant) non-small cell lung cancer. In anembodiment, the method can include administering the compound of thepresent invention to a subject (e.g., a mammal or a warm blooded animal)having pancreatic cancer. In an embodiment, the method can includeadministering the compound of the present invention to a subject (e.g.,a mammal or a warm blooded animal) having breast cancer that expressesHER-2. In an embodiment, the subject is a human.

The methods have been described with reference to “a compound of thepresent invention.” In the description of the methods of the invention,the phrase “a compound of the present invention” can be replaced by thephrase “a pharmaceutically acceptable salt of a compound of the presentinvention”, the phrase “a pharmaceutical composition including acompound of the present invention” or the phrase “a pharmaceuticalcomposition including a pharmaceutically acceptable salt of a compoundof the present invention” to describe additional embodiments of thepresent methods.

The compound of the present invention can be administered at a dosesufficient to provide a therapeutically effective level at a tumor cellin the subject. It is recognized that the total amount of compound ofthe present invention administered as a unit dose to a subject willdepend upon the type of pharmaceutical composition being administered.It should be apparent to a person skilled in the art that variations maybe acceptable with respect to the therapeutically effective dose andfrequency of the administration of compound of the present invention inthis embodiment of the invention. The amount of the compound of thepresent invention administered will be inversely correlated with thefrequency of administration. Hence, an increase in the concentration ofcompound of the present invention in a single administered dose, or anincrease in the mean residence time in the case of a sustained releaseform of compound of the present invention, generally will be coupledwith a decrease in the frequency of administration.

The actual dose of the compound of the present invention will depend ona variety of factors that may be specific to the subject undergoingdosing. These factors should be taken into consideration whendetermining the therapeutically effective dose of compound of thepresent invention and frequency of its administration. For example, theeffective dose can depend on the species, age, weight, or general healthof the subject; the severity of the cancer or tumor growth; the size andlocation of the tumor in which an effective amount of agent must beachieved; the frequency and duration of dosing; the type of formulationadministered; and the like. Generally, a higher dosage is preferred ifthe disease or disorder is more severe.

Methods of Making the Present Compounds

Scheme 1 illustrates an embodiment of a process for making compounds ofthe present invention.

Pharmaceutical Compositions

The present compound can be in a pharmaceutical composition thatincludes a pharmaceutically acceptable carrier mixed with the compoundof the present invention and other components in the pharmaceuticalcomposition. As used herein, the term “pharmaceutically acceptablecarrier” refers to a carrier that is conventionally used to facilitatethe storage, administration, and/or the healing effect of the ananticancer or antiproliferative agent. A carrier may also reduce anyundesirable side effects of the agent. A suitable carrier should bestable, i.e., incapable of reacting with other ingredients in theformulation. It should not produce significant local or systemic adverseeffect in recipients at the dosages and concentrations employed fortreatment. Such carriers are generally known in the art.

In an embodiment, the pharmaceutical composition can include:

Tablet mg/tablet Compound NRC-2694 or a salt thereof 50 Lactoseanhydrous (USP) 156 Microcrystalline cellulose (Avicel pH102) 15 Sodiumlauryl sulfate 5 Sodium starch glycolite 10 Povidone K-30 3 Hydroxypropyl cellulose (LH-11) 10 Magnesium stearate 1

The agent of the present invention can also be formulated in asustained-release form to prolong the presence of the pharmaceuticallyactive agent in the treated mammal, generally for longer than one day.Many methods of preparation of a sustained-release formulation are knownand are disclosed in Remington's Pharmaceutical Sciences (18th ed.; MackPublishing Company, Eaton, Pa., 1990).

Articles and Methods of Manufacture

The present invention also includes an article of manufacture providingan agent for administration to a subject having a tumor or to a tumorcell. The article of manufacture can include a container which containsa composition (e.g., tablet) suitable for the present method. Thearticle of manufacture further includes instructions in the form of alabel on the container and/or in the form of an insert included in a boxin which the container is packaged, for the carrying out the method ofthe invention. The instructions can also be printed on the box in whichthe container is packaged. The instructions contain information such assufficient dosage and administration information so as to allow thesubject or a worker in the field to administer the present compound orpharmaceutical composition. It is anticipated that a worker in the fieldencompasses any doctor, nurse, technician, spouse, or other caregiverwho might administer the agent. The agent can also be self-administeredby the subject.

According to the invention, the present compound of formula I (e.g.,compound of formula IA) can be used for manufacturing an composition ormedicament including the compound and suitable for oral or parenteraladministration. The invention also relates to methods for manufacturinga composition or medicament including a compound of formula I (e.g.,compound of formula IA) suitable for oral or parenteral administration.For example, a tablet can be manufactured in several ways, usingconventional techniques. A liquid composition can be manufactured bydissolving an agent in a suitable solvent, such as water, at anappropriate pH, including buffers or other excipients, for example, toform a solution.

The present invention may be better understood with reference to thefollowing examples. These examples are intended to be representative ofspecific embodiments of the invention, and are not intended as limitingthe scope of the invention.

EXAMPLES Example 1 Preparation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(IA) i) Preparation of4-Chloro-6-[3-(4-morpholinyl)propoxy-4-quinazoline (IIIa)

Into a clean and dried 5-Litre four necked round bottomed flask equippedwith a mechanical stirrer, reflux-condenser, pressure equalizingaddition funnel, and thermometer socket were charged chloroform (3000ml), dimethyl formamide (30 ml) followed by 7-methoxy-6-(3-morpholinopropoxy)-3,4-dihydro-quinazolin-4-one (IIa) (150 g), obtained accordingto the process given in Example-1 of PCT international applicationpublished as WO2005/070909A1. Oxalyl Chloride (120 g) was slowly addedand the reaction mass was heated to reflux temperature and maintained atreflux temperature for about 5 hours. Reaction was found to be completedby HPLC test. The solvent chloroform and excess oxalyl chloride weredistilled off by applying mild vacuum. The reaction mass was cooled toabout 40° C. and added chloroform (300 ml) and again distilled out thesolvent by applying mild vacuum. The reaction mixture was cooled to roomtemperature and acetonitrile (3000 ml) was added and stirred for 10-15minutes and kept under nitrogen atmosphere to proceed to the next step.

ii) Preparation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(IA)

Into a 5-Litre four necked round bottomed flask equipped with amechanical stirrer, reflux-condenser and thermometer socket containingthe chloro compound in acetonitrile from the above step-(i); 3-ethynylaniline (69 g) was added slowly in about 10-15 minutes and the reactionmass was heated to reflux temperature and maintained at refluxtemperature for about 4 hrs. The reaction was found to be completed byHPLC test. Then the reaction mass was cooled to 25-35° C. and filtered,washed the cake with acetonitrile (500 ml) and dried the cake.

The above dried crude compound was taken into a another 5 liter roundbottomed flask and charged water (2500 ml) and slowly raised thetemperature to 60-65° C. and was adjusted the pH of the reaction mass to10-12 with dilute sodium hydroxide solution. The solid product separatedwas filtered and washed with water and dried at 70-75° C. to get 173.0 gof N-(3-ethynylphenyl)-6-(3-morphilinepropoxy)-7-methoxy-4-quinazolamine as a off-white solid.

iii) Recrystallisation of Preparation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminefrom Toluene.

Into a 5-Litre four necked round bottomed flask equipped with amechanical stirrer, reflux-condenser and thermometer socket were chargedtoluene (3750 ml), followed by N-(3-ethynylphenyl)-6-(3-morpholinopropoxy)-7-methoxy-4-quinazolinamine (50 g) obtained by the processdescribed in the above given example-(1). The reaction mixture washeated to 90-95° C., so that the solid completely dissolved. Then carbontreatment was given and filtered. The filtrate was cooled to 25-35° C.,maintained for about 1 hour and filtered and dried the material to get40.15 g ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamineas a white crystalline solid.

mp: 185-187° C.

Purity: 99.72% (HPLC)

IR (KBr) (cm⁻¹): 3280.9, 2954.6, 2810.3, 1620.1, 1604.2, 1572.1, 1527.7,1505.2, 1484, 1430.5, 1388.2, 1247.5, 1211.2, 1140.3, 1110.4, 1010.3,953.4, 859.6, 784.2 Cm⁻¹

¹HNMR (300 MH_(z); DMSO-d₆): 9.57 (s, 1H); 8.48 (s, 1H); 7.99 (s, 1H);7.86 to 7.92 (d, 2H); 7.34 to 7.44 (t, 1H) 7.18 to 7.21 (s, 2H); 4.15 to4.21 (t, 4H); 3.92 (s, 3H) 3.5 to 3.6 (t, 4H); 2.4 to 2.52 (m, 5H); 1.95to 2.01 (m, 2H).

Mass: 419.4 (M+1)

Example 2 Recrystallisation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminefrom acetonitrile

Into a two liter three necked round bottomed flask equipped with amechanical stirrer, reflux-condenser and thermometer socket were chargedacetonitrile (1000 ml), followed byN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(25 g) obtained from the process described in the above givenExample-(1). The reaction mass was slowly heated to 65-70° C., so thatthe solid material completely dissolved and carbon treatment was givenand filtered the reaction mass. The filtrate was transferred intoanother round-bottomed flask and slowly cooled to 10-15° C. andmaintained for 30 minutes at that temperature. The mass was filtered andafter washing the cake with chilled acetonitrile dried to get 20.50 g ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamineas a white crystalline solid.

mp: 186-187° C.;

Purity: 99.68% (HPLC)

Example 3 Recrystallisation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminefrom Ethyl acetate

Into a three liter three necked round bottomed flask equipped with amechanical stirrer, reflux-condenser and thermometer socket were chargedethyl acetate (2000 ml), followed byN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(25 g) obtained from the process described in the above givenExample-(1). The reaction mass was slowly heated to 65-70° C., so thatthe solid material completely dissolved and carbon treatment was givenand filtered the reaction mass. The filtrate was transferred intoanother round-bottomed flask and slowly cooled to 10-15° C. andmaintained for 30 minutes at that temperature. The crystalline mass wasfiltered and after washing the cake with chilled ethyl acetate dried toget 20.95 g of N-(3-ethynlphenyl)-6-(3-morpholinopropoxy)-7-methoxy-4-quinazolinamine as a white crystalline solid.

mp: 185-187° C.

Purity: 99.7% (HPLC)

Example 4 Preparation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminemono hydrochloride (IA monohydrochloride)

Into a 500 ml three necked round bottomed flask equipped with amechanical stirrer, reflux-condenser, thermometer socket etc. chargedIsopropyl alcohol (250 ml), followed byN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine(5 g), obtained from the process given in Example-1. The temperature ofthe reaction mass was raised to 65-70° C. so that all the solid materialdissolves and carbon treatment was given and filtered. The filtrate wascooled to about 55 to 60° C. and to this one mole equivalent of HCl-gasdissolved in isopropyl alcohol solution was added when the monohydrochloride salt separated out. The reaction mass was maintained atreflux temperature for about 2 hrs and then cooled to room temperatureand filtered and dried to get 5.1 g. of N-(3-ethynylphenyl)-6-(3-morpholino propoxy)-7-methoxy-4-quinazolinamine monohydrochloride as a white crystalline substance.

Purity: 99.8% (HPLC)

HCl content (chemical): 8.19% (Theoretical value: 8.01%)

IR (KBr) (cm⁻¹): 3407, 3305, 3259.5, 2934, 2619, 1625.9, 1593.8, 1579.9,1530.8, 1512, 1476.9, 1392.2, 1356.8, 1282.1, 1242.1, 1207.9, 1141.3,1100.8, 1076.1, 1042.1, 1026.5, 1011.5, 957.7, 941.5, 922.1, 857.3, 852,838.1, 796, 782.4.

Example 5 Preparation ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminedi hydrochloride (IA dihydrochloride)

Into a 500 ml three necked round bottomed flask equipped with amechanical stirrer, reflux-condenser and thermometer socket were chargedIsopropyl alcohol (250 ml), followed byN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinamine5 g), obtained from the process given in Example-1. The temperature ofthe reaction mass was raised to 65-70° C. so that all the solid materialdissolves. Carbon treatment was given and filtered. The filtrate wascooled to about 55 to 60° C. and to this two moles equivalent of HCl-gasdissolved in isopropyl alcohol solution was added when thedihydrochloride salt separated out. The reaction mass was maintained atreflux temperature for about 2 hrs and then cooled to room temperatureand filtered and dried to get 5.5 g. ofN-(3-ethylnylphenyl)-7-methoxy-6-[3-(4-morpholinyl)propoxy]-4-quinazolinaminedi hydrochloride as a white crystalline substance.

Purity: 99.78% (HPLC)

HCl content (chemical): 14.9% (Theoretical value: 14.83%)

IR (KBr) (cm⁻¹): 3406.8, 3194.1, 2942.7, 2681.9, 2623.6, 1633.7, 1566.2,1528.6, 1512.5, 1438.6, 1359.6, 1282.3, 1218.3, 1157.1, 1132.7, 1105.9,1075.6, 1001.9, 942.1, 875.3, 816.1, 787.2

Example 6 In Vitro Kinase Profiling of the Compound of Formula (IA)

NRC-2694 (the compound of formula IA) was found to possess activityvarious kinases from a panel of 80 kinases by the method of KinomeScan™of Ambit Biosciences Corp, San Diego, Calif. The kinases wereABL1(F317L), ABL1(H396P), ABL1(Q252H), ABL1(Y253F), ADCK3, ADCK4, ALK,ARK5, AXL, BLK, BRSK2, CDK7, CIT, CLK4, CSNK1D, CSNK1E, DAPK1, DAPK3,DCAMKL3, DMPK, EGFR, EGFR(E746-A750DEL), EGFR(G719C), EGFR(G719S),EGFR(L747-E749DEL, A750P), EGFR(L747-S752 DEL, P753S),EGFR(L747-T751DEL, Sin), EGFR(L858R), EGFR(L861Q), EGFR(S752-I759DEL),EPHA5, EPHA6, ERBB2, ERBB4, ERK3, ERK4, FGR, FLT3(D835H), FLT3(D835Y),FLT3(ITD), FLT3(N841 I), FRK, GAK, GCN2(KIN.DOM.2, S808G), HCK, IRAK3,JAK1(KIN.DOM.1), KIT, KIT(D816V), KIT(V559D), KIT(V559D,T670I), LCK,LOK, LTK, LYN, MAP3K3, MAP4K4, MAP4K5, MET, MINK, MKNK1, MKNK2, MST4,MYLK2, PDGFRB, PIM3, PKN2, PRKD1, PRKD2, PRKD3, PRKG1, RIPK2,RPS6KA4(KIN.DOM.2), SIK2, SLK, SNARK, SRC, STK36, TNIK, TNNI3K,TYK2(KIN.DOM.2).

Kinase assays: Assays were performed as described in Fabian et al.(2005) Nature Biotechnology, vol. 23, p. 329. Kinase-tagged T7 phagestrains were grown in parallel in 24 or 96-well blocks in an E. colihost derived from the BL21 strain. E. coli were grown to log-phase andinfected with T7 phage from a frozen stock (multiplicity of infection˜0.1) and incubated with shaking at 32° C. until lysis (˜90 minutes).The lysates were centrifuged and filtered to remove cell debris.Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The liganded beads were blocked with excessbiotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05%Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specificphage binding. Binding reactions were assembled by combining phagelysates, liganded affinity beads, and test compounds in 1× bindingbuffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT).

Test compounds were prepared as 40× stocks in DMSO and diluted into theaqueous environment. 2.5% DMSO was added to control assays lacking atest compound. All reactions were performed in polystyrene 96-wellplates that had been pretreated with blocking buffer in a final volumeof 0.04 ml. The assay plates were incubated at room temperature withshaking for 1 hour, long enough for binding reactions to reachequilibrium, and the affinity beads were washed four times with washbuffer (1×PBS, 0.05% Tween 20, 1 mM DTT) to remove unbound phage. Afterthe final wash, the beads were resuspended in elution buffer (1×PBS,0.05% Tween 20, 2 μM non-biotinylated affinity ligand) and incubated atroom temperature with shaking for 30 minutes. The phage titer in theeluates was measured by quantitative PCR.

Results

The activity profile results are shown in charts 1.1-1.6. Significantvalues are shaded. The kinases against which NRC-2694 (the compound offormula IA) is most active is indicated as (++) and where it has shownvery little inhibiting effect is indicated as (−−) in the followingtable.

Kinase Activity Trends-NRC2694 (IA) (++) (−−) ALK, AKT1, AKT2, BLK,ASK1, AURKA₁, CDK7, BMPR¹A, BMPR², CIT CDK5, CDK8, EGFR EPHA2, ERK2,ERK-3 FGFR², FGFR3, ERK-4 GSK3B, LMK2, FLT MARK4, GAK, MEK3, MLK1, MST1,LCK MUSK, MYO3B, LOK p38, PCTK, P1K3CA, MKNK PKAC, PRKX, PDGFR-B RAF-1,RET, RPS6KA, PRKD SRMS, STK16, SLK TRKA, ULK, RIPK2 YANK, ZAP70

Example 7 Anticancer Activity of the Compound of Formula (IA) inPancreatic Cancer Models

In vivo anticancer activity of NRC-2694 (the compound of formula IA) forthe treatment of Pancreatic Cancer in nude mice xenografts (Athymic nudemice).

Methodology

Athymic nude mice were implanted with 10×10⁶ pancreatic cancer cells MIAPaCa-2 (ATCC #CRL1420). After tumor formation (˜0.3-0.05 cm³) mice weretreated with study compounds with or without Gemcitabine. Tumor volumeand animal activity were measured at regular intervals. At thetermination of the experiment tumors were collected and fixed in 10%buffered formaldehyde for 12 hours and processed for paraffin embeddingfollowed by sectioning. Tumor sections were immunoprobed for VEGF andKi67 and percent expression determined with respect to controls.

Study Design Group # Study Drugs Dose (mg/kg) Route Number of Animals 1Control 0 Oral 5 Male + 5 Females 2 NRC 2694 20 Oral 5 Male + 5 Females3 Erlotinib 50 Oral 5 Male + 5 Females 4 Gemcitabine 120 i.p. 5 Male + 5Females 5 NRC 2694 + 20 + 120 Oral + ip 5 Male + 5 Females Gemcitabine 6Erlotinib + 50 + 120 Oral + ip 5 Male + 5 Females GemcitabineResults

From the study conducted it was observed that NRC-2694 (the compound offormula IA) did cause the regression of pre-established subcutaneouspancreatic tumors in nude mice. Tumor regression caused by NRC2694 (thecompound of formula IA) was similar to Erlotinib and Gemcitabinetreatments. However, addition of Gemcitabine caused a greater thanadditive effect. Erlotinib hydrochloride (TARCEVA®) is an approvedstandard drug for the first-line treatment of patients with locallyadvanced, unresectable or metastatic pancreatic cancer, in combinationwith Gemcitabine. Gemcitabine (GEMZAR®) is indicated for the first linetreatment for patients with locally advanced non-resectable ormetastatic adenocarcinoma of the pancreas.

The tumor regression caused by treating with compound IA was superior tothat caused by Erlotinib and Gemcitabine individually as well as incombination (FIG. 1). This observation was further confirmed inImmunohistochemical analysis (FIG. 2).

Example 8 Anticancer Activity of the Compound of Formula (IA) in HER-2Positive Breast Cancer

In vivo anticancer activity of NRC-2694 (the compound of formula IA) forthe treatment of HER-2 Positive Breast Cancer in SCID mice xenografts.

Methodology

Animals were subcutaneously implanted with 2×10⁶ cells. After thedevelopment of observable tumors drug treatment was initiated. As perIRB regulations no animals were allowed to bear tumors greater than 15mm. Cell line: BT474/HTB20 human breast cancer cell line.

Study Design Group # Study Drugs Dose (mg/kg) Route Number of Animals 1Control 0 Oral 5 Male + 5 Females 2 NRC 2694 10, 20 and 40 Oral 5 Male +5 Females (once a day) 3 Lapatinib 30 and 100 Oral 5 Male + 5 Females(Twice a day)Results

NRC-2694 (the compound of formula IA) demonstrated a dose dependentincrease in anti tumor activity in HER-2 positive Breast cancer model.The tumor regression observed with NRC-2694 (the compound of formula IA)treated animals was superior to that of Lapatinib treated animals (FIG.3). Lapatinib (TYKERB®) is a kinase inhibitor indicated in combinationwith Capecitabine for the treatment of patients with advanced ormetastatic breast cancer whose tumors over express HER2 and who havereceived prior therapy including an anthracycline, a taxane andtrastuzumab. NRC-2694 (the compound of formula IA) was superior toLapatinib, the established treatment option for HER2 expressing tumors,which was significant and unexpected.

Example 9 Anticancer Activity of the Compound of Formula (IA) inErlotinib/Gefitinib Resistant NSCLC Model

In vivo activity of NRC-2694 (the compound of formula IA) for thetreatment of Erlotinib/Gefitinib resistant Non Small Cell Lung Cancer(NSCLC) in nude mice xenografts (athymic nude mice).

Methodology

Cells: H1975. Athymic nude mice, 5-8 weeks of age and weighing 20 to 25g, were housed in the animal facility. H1975 cells were harvested fromexponentially growing cultures, detached by brief trypsinization, washedtwice in cold HBSS, resuspended in ice-cold HBSS, and implantedsubcutaneously (3×10⁶ cells per mouse) into the dorsal hind flank.Treatment with the study drugs was initiated only after the tumor volumereached to 200-300 mm³.

Study Design Study Dose Group # Drugs (mg/kg) Route Number of Animals 1Control 0 Oral 5 Male + 5 Females 5 NRC 2694 10 Oral 5 Male + 5 Females6 NRC 2694 20 Oral 5 Male + 5 Females 7 NRC 2694 40 Oral 5 Male + 5Females 8 Erlotinib 100 Oral 5 Male + 5 FemalesResults

NRC 2694 (the compound of formula IA) demonstrated significantanticancer activity in animals implanted with Erlotinib/Gefitinib NSCLCresistant cells compared to Erlotinib or control. It showed dosedependent decrease in the tumor volume. Tumor remission was continued inanimals treated with NRC 2694 (the compound of formula IA) even afterwithdrawing the study treatment (FIG. 4).

Drug resistance for treatment of non-small cell lung cancer (NSCLC) byapproved drugs like Erlotinib (TARCEVA®) and Gefitinib (IRESSA®) is aclinical challenge posed and observed in recent times (Clin. Cancer.Res. 2006, 12(19): 5764-69; PLOS Medicine, March 2005, Online edition0225-0235). The significant superior activity (250% reduction in tumorvolume) observed with NRC-2694 (the compound of formula IA) wasunexpected.

Example 10 NRC-2694 is Less Toxic Than Erlotinib in Swiss Albino Mice

This study determined that NRC-2694 (the compound of formula IA) is lesstoxic than erlotinib in Swiss albino mice. The maximum tolerated dose(MTD) of NRC-2694 is about twice that of erlotinib. The MTD of NRC-2694is the same in mice and rats.

Materials and Methods

The method followed was as per the guidelines of FDA (Aug. 26, 1996).

Swiss Albino Mice

20 male and 20 female Swiss albino mice were randomly divided into fourgroups (G1-G4). Each group was 5 male and 5 female Swiss albino mice.Groups G2, G3 and G4 were treated with NRC-2694 monohydrochloridethrough oral route at the dose levels of 250, 500 and 1000 mg/kg bodyweight respectively. G1 was treated with placebo and served as acontrol. The oral formulation included 25 mg/mL of NRC-2694 that wasadministered in doses of 10 mL/kg body weight (b.wt) to each animal.Animals from the low dose group (G2-treated with 250 mg/kg b.wt) wereadministered the oral formulation as a single dose. The mid dose groupanimals (G3-treated with 500 mg/kg b.wt) were administered the doseformulation twice with a gap of approximately 1 hour. Animals from highdose group (G4-treated with 1000 mg/kg b.wt) received the doseformulation four times with a gap of approximately one hour.

Dose formulation analysis for test substance revealed 105.1, 105.74 and105.76% recovery whereas dose formulation of placebo substance revealed0% recovery of active ingredient.

At the end of observation period of 14 days, all surviving mice weresacrificed and subjected to gross pathological examinations.

Wistar Rats

20 male and 20 female Wistar rats were randomly divided in to fourgroups and dosed as described above for the mice. Dose formulationanalysis for test substance revealed 101.0, 102.84 and 103.56% recoverywhereas dose formulation of placebo substance revealed 0% recovery ofactive ingredient.

Results

Mice: No mortality was observed in mice from the control as well as thetreatment groups. Clinical signs of toxicity such as mild lethargy andpiloerection were observed in high dose group after dosing during days10 to 14. No significant alterations were observed in mean body weightand percent body weight when compared with their respective controlgroups. Gross pathological examination after day 14 did not reveal anylesions of pathological significance.

Rats: No mortality was observed in rats from the control as well as thetreatment groups. In the high dose treated group (G4-1000 mg/kg bodywt.) clinical signs of toxicity such as mild to severe lethargy (10/10),piloerection 10/10), chromodacryoorhea (10/10), weakness (1/10) andptosis (4/10) were observed. Statistically significant reduction wasobserved in body weight and percent body weight change of mid and highdose treated group of rats on days 7 and 14. Reduction observed in thebody weight and percent body weight change in high dose treated animalswas considered as an effect of oral administration of NRC-2694monohydrochloride. Gross pathological examination did not reveal anylesions of pathological significance, except minimal uterus distensionin two female rats. This lesion was physiological or cyclic in natureand could be considered as a spontaneous finding.

Conclusions

Mice: Based on the findings of this study, it was concluded that thetest substance NRC-2694 monohydrochloride did not produce any toxicsigns or mortality when administered through oral gavage at the doselevel of 250 mg/kg body weight (low dose) and 500 mg/kg (mid dose). At1000 mg/kg (high dose) the NRC-2694A blend produced signs of lethargyand piloerection in all animals without any mortality. The MTD ofNRC-2694 monohydrochloride by acute oral gavage in Swiss albino mice was1000 mg/kg body weight under the condition and the procedures followedin the present study.

Under our laboratory condition, the MTD of Erlotinib HCl was recorded tobe 500 mg/kg, when given orally to Swiss albino mice. Thus, NRC-2694 isadvantageously significantly less toxic than Erlotinib HCl in mice.

Rats: Based on the findings of this study, it was concluded that thetest substance NRC-2694A blend did not produce any major toxic signs ormortality when administered through oral gavage at the dose level of 250mg/kg body weight (low dose) and 500 mg/kg body weight (mid dose).However, at the dose level of 1000 mg/kg body weight (high dose), theNRC-2694A blend produced marked toxic signs with reduction in bodyweight but devoid of any pathological lesions and mortality. Based onthe above study, the MTD of NRC-2694A blend by acute oral gavage inWistar rats is considered as 1000 mg/kg body weight under the conditionand the procedures followed in the present study.

Example 11 NRC-2694 is Less Toxic Than Erlotinib in Beagle Dogs

This study determined that NRC-2694 (the compound of formula IA) is lesstoxic than erlotinib in beagle dogs. The no observed adverse effectlevel (NOAEL) of NRC-2694 is about twice that of erlotinib.

Materials and Methods

The methods followed were as per CPMP/SWP/1042/99 (July 2000) and ICHS3A (March (1995)) guidelines. Prior to the 30 day repeated dose study,a dose range finding study was conducted, which included single dose MTDstudy followed by a 10 day repeated dose study. For the single dose MTDstudy, two dogs (1 male and 1 female) of beagle breed, obtained fromMarshall's Farm, China, were used. The route of dosing was oral, bygelatin capsule. Based on the results of the dose range finding study,doses for the 30 days study were selected.

The 30 day repeated dose study employed 12 male and 12 female dogs(Marshall's Farm, China). The dogs were acclimatized for a minimumperiod of 2 weeks and randomly divided into four main groups (G1-G4),each group comprising of 3 dogs per sex. The animals were dosed orallywith capsules of NRC-2694A for a period of 30 consecutive days at doselevels of 10 mg/kg b.wt/day (G2-low dose), 20 mg/kg b.wt/day (G3-middose) and 40 mg/kg b.wt/day (G4-high dose). However, the high dose wassubsequently decreased to 30 mg/kg b.wt/day. The control group (G1-0mg/kg b. wt/day) animals were dosed by oral capsule with placebo.

Each dog was observed for visible signs of reaction once daily and formortality and morbidity twice daily throughout the study period. Thestudy also tracked body weight (weekly), food consumption (daily),opthalmological status (start and end), hematological and biochemicalanalyses (start and end), toxicokinetics analysis of plasma (start andend). At the end of the study, all dogs were euthanized by a humane andaccepted procedure, subjected to a gross post-mortem examination, organweights (absolute and relative) were determined, and histopathologicalexamination was carried out.

Results

No symptoms of toxicity observed in the low dose (G2) and control (G1)groups. The toxicokinetic parameters viz. Lambda z, HL Lambda z,T_(max), C_(max), AUC₀₀ of NRC-2694 monohydrochloride on Day 1 wascalculated separately for male and female dogs by using WinNonlinversion 5.2 software. There was a dose-dependent change in the C_(max)of NRC-2694 monohydrochloride at the three dose levels in both male andfemale dogs. The median T_(max) for attaining C_(max) at three doselevels (10, 20 and 40 mg/kg) on day 1 were 2, 3, 2 hr in male and 2, 4,4 hr in female, respectively. The median T_(max) for attaining C_(max)at two dose levels (10 and 20 mg/kg) on day 30 were 2 and 3 hr in maleand 2 and 4 hours in female, respectively.

Conclusion

Based on the results of this study, it was concluded that the noobserved adverse effect level (NOAEL) of NRC-2694 monohydrochloride inbeagle dogs dosed over a period of 30 days is 10 mg/kg b.wt/day whenadministered orally by capsule. The corresponding value for ErlotinibHCl is 5 mg/kg b.wt/day. Thus, NRC-2694 is advantageously significantlyless toxic than Erlotinib HCl in beagle dogs.

Example 12 NRC-2694 is Not Mutagenic in the Ames Test

This study determined that NRC-2694 (the compound of formula IA) is notmutagenic in the Ames test.

Materials and Methods

The present study was conducted by the direct plate incorporation methodwith five tester strains of Salmonella typhimurium (TA 98, TA 100, TA102, TA 1535 and TA 1537). NRC-2694 was tested at the doses of 1.2500,0.3955, 0.1251, 0.0396 and 0.0125 mg/plate. Simultaneously, negativecontrol cultures received DMSO and the respective positive controlsreceived the mutagens 2-Amino anthracene, 2-Nitrofluorene, Sodium azide,9-Aminoacridine and Mitomycin C (Sigma, St. Louis). In order to studythe role of metabolic activation, cultures were incubated both with andwithout S9 mixture. The induction of Histidine positive colonies wascomputed and results were statistically treated for comparison.

Results and Conclusion

The study indicated lack of statistically significant induction of Hisrevertant colonies using NRC-2694 in any of the tester strains eitherwith or without S9 addition in the culture when compared to positive andnegative controls. Based on the above results, it was concluded that theNRC-2694 monohydrochloride was non-mutagenic according to the Amesbacterial reverse mutation assay.

Example 13 NRC-2694 Lacks Neurotoxicity in the Functional ObservationalBattery

This study determined that NRC-2694 (the compound of formula IA) showedno evidence of neurotoxicity toward the central and peripheral nervoussystem in Wistar rats using the functional observations battery (FOB)test.

Materials and Methods

The study was conducted in accordance with “S7A Safety PharmacologyStudies for Human Pharmaceuticals, ICH” July 2001, and as per this studyprotocol. The FOB assessed the effect of NRC-2694 monohydrochloride onthe central and peripheral nervous systems in Wistar rats. Rats that hadbeen fasted for 16 to 18 hours (access to water was not interrupted)were dosed with the compound or water. The rats were administered thecompound in an aqueous formulation at three levels, 75, 125 and 250mg/kg b.wt, as a single oral dose by gavage. The control group receivedwater. The rats were subjected to the FOB 60-120 min. after drugadministration.

Animals were subjected to the FOB one at a time except for the multipleactivity cage where four animals were placed in four different cagessimultaneously. Once complete, the animals were returned to their homecage and the cage placed back on the stand.

Results and Discussion

The functional observational battery tests carried out did not show anyadverse effects at the tested doses. NRC-2694A was well tolerated at thegiven doses having no influence on any of the parameters of the FOB60-120 minutes post treatment.

The FOB can be employed to assess multiple neurobiological domainsincluding neuromuscular (weakness, in coordination, gait and tremor),sensory (audition, vision and somatosensory), and autonomic (pupilresponse and salivation) functions. The measures of a neuro behavioralscreening battery have been divided into specific functional domains.For example, Lacrimation, salivation, pupil response, palpebral closure,defecation and urination are measures of some aspects of the autonomicsystem. Similarly the neuro muscular domain can be assessed based on thegait/mobility score, landing foot splay, grip strength and rightingreflex; sensorimotor domain based on response to tail pinch andclick/touch/approach response; CNS excitability domain based on ease ofremoval and handling of the animal, clonic/tonic movements, arousal andvocalization; CNS activity domain based on home cage posture, palpebralclosure, rearing and motor activity and finally the physiological domainis assessed based on the body weight, body temperature and piloerection.

Example 14 NRC-2694 Lacks Toxicity Against Respiratory Function

This study determined that NRC-2694 (the compound of formula IA) lackstoxicity against respiratory function in Wistar rats.

Materials and Methods

This study evaluated the effect of NRC-2694A monohydrochloride onrespiratory function in conscious Wistar rats following administrationof a single dose using a whole body plethysmograph. The study wasconducted in accordance with “S7A Safety Pharmacology Studies for HumanPharmaceuticals, ICH” issued July 2001. The study used a parallel designwith one vehicle and three test item treated groups. NRC-2694monohydrochloride was administered orally at the doses of 75, 125 and250 mg/kg body weight. Male Wistar rats were placed in plethysmographychambers and baseline respiratory parameters were recorded for 30minutes. Following drug administration respiratory parameters wererecorded continuously for 3 hours.

Results

At the tested doses NRC-2694 monohydrochloride did not influence any ofthe observed parameters: respiratory rate, tidal volume, minute volume,and Penh.

Example 15 NRC-2694 Lacks Toxicity Against Cardiovascular Function

This study determined that NRC-2694 (the compound of formula IA) lackstoxicity against cardiovascular function in beagle dogs.

Materials and Methods

This study evaluated the effect of orally administered NRC-2694monohydrochloride on cardiovascular parameters in telemetered maleBeagle dogs. The study was conducted in accordance with ICH “S7A SafetyPharmacology Studies for Human Pharmaceuticals, Note for Guidance onSafety Pharmacology Studies for Human Pharmaceuticals, July 2001”. Thedoses tested were 0, 15, 30 and 60 mg/kg b.wt. given orally by gelatincapsule. Four male Beagle dogs, each implanted with a TL1IM2-D70-PCTtransmitter (Data Sciences International, USA), were used for the study.During the study, the dogs were administered 4 oral treatmentsrepresenting placebo and all test substance treatment groups with athree day (˜72 hour) wash-out period between each administration, usinga Latin square cross-over design.

On dosing days, the telemetered dogs were observed once beforecommencement of data collection, continuously through 2 hours post-doseremotely and ˜8 hours post-dose for changes in behaviour. Cardiovascularparameters (systolic, diastolic and mean blood pressure, pulse pressureand heart rate) were recorded continuously for ˜60 minutes before dosingand up to 24 hours after dosing. Systolic, diastolic and mean bloodpressures, pulse pressure and heart rate were obtained from the femoralartery waveform. Electrocardiograms were recorded continuously for ˜60minutes before dosing and up to 24 hours after dosing.Electrocardiograms were obtained from subcutaneously placedbio-potential leads in a Lead II configuration. Heart rate and QTintervals were determined from the ECG's.

Results

When administered orally to male Beagle dogs, NRC-2694 monohydrochloridehad no significant negative effects on cardiovascular parameters(systolic, diastolic and mean blood pressure, pulse pressure, heartrate, or QT and corrected QT (QTcf) intervals) at the doses tested. Theno observed effect level was 60 mg/kg b.wt.

Example 16 NRC-2694 is Safe in a Test of Cardiac Risk Assessment

This study determined that NRC-2694 (the compound of formula IA) is safein a recombinant cell system used as an index of QT intervalprolongation for cardiac risk assessment.

Materials and Methods

The effect of NRC-2694 monohydrochloride on the potassium selective Ikr(tail) current (the rapid component of the delayed rectifier current)was investigated using Chinese hamster ovary (CHO) cells stablytransfected with hERG (human ether-a go-go related gene) employing thewhole cell patch-clamp technique. Blockade of the Ikr potassium currentis considered to constitute an index of QT interval prolongation forcardiac risk assessment. The test was conducted according to S7B: TheNonclinical Evaluation of the Potential for Delayed VentricularRepolarization (QT Interval Prolongation) By Human Pharmaceuticals. ICHMay 2005

This CHO cells stably transfected with hERG were from Flyion GmBH(Germany). Stock cultures of hERG-TRex-CHO cells were stored as frozenpermanents in liquid nitrogen. The test compound was evaluated at 10 μMin 0.1% DMSO (v/v). Propafenone (Sigma, St. Louis) at 10 μM was thepositive control. The vehicle control was 0.1% (v/v) DMSO in ECsolution.

The selection medium was F-12 (Ham)+GlutaMAX, Ph 7.2, 10% FBS,Penicillin (100 U/mL), Streptomycin (100 μg/mL), Blasticidin S HCL (30μg/mL) and Hygromycin B (400 μg/mL). The growth medium was F-12(Ham)+GlutaMAX, 10% FBS, Penicillin (100 U/mL) and Streptomycin (100μg/mL). The induction medium was F-12 (Ham)+GlutaMAX, 10% FBS,Penicillin (100 U/mL) and Streptomycin (100 μg/mL) and Doxycycline (3μg/mL). The cell dissociation solution was 0.05% Trypsin-EDTA. Theextracellular solution (and robot medium) was NMDG 145 mM, KCl 5 Mm,MgCl₂(H₂O)₆ 1 Mm, HEPES 10 mM, Glucose 10 mM, at pH 7.4. Theintracellular solution was KMeSO₃ 115 mM, MgCl₂(H₂O)₆ 5 mM, HEPES-KOH 10mM, EGTA 5 mM, K₂ATP 5 mM, at pH 7.2.

Results and Discussion

The test compound was evaluated at 10 μM where it showed ˜42% inhibitionof hERG current. This suggests that NRC-2694, in the experimentalcontext of this study, was an inhibitor of the hERG channel. Thepositive control, propafenone, caused ˜69% inhibition at 10 μM,indicating the sensitivity of the test system. The tail current mainlyreflects the open channel state and is the appropriate index for hERGchannel evaluation. Inhibition of the hERG channel can lead to delayedrepolarization, which can manifest itself as a prolongation of theQT-interval.

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

All publications and patent applications in this specification areindicative of the level of ordinary skill in the art to which thisinvention pertains.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

CHART 1.1 In vitro kinase profiling of NRC-2694 Kinase Target NRC-2694Descriptive Gene Symbol Ambit Gene Symbol Clone ID (Primary) % Ctrl @10000 nM AAK1 AAK1 AAK1 38 ABL1 ABL1 ABL1 67 ABL1 (E255K) ABL1 (E255K)ABL1 (E255K) 80 ABL1 (F317I) ABL1 (F317I) ABL1 (F317I) 48 ABL1 (F317L)ABL1 (F317L) ABL1 (F317L) 33 ABL1 (H396P) ABL1 (H396P) ABL1 (H396P) 34ABL1 (M351T) ABL1 (M351T) ABL1 (M351T) 37 ABL1 (Q252H) ABL1 (Q252H) ABL1(Q252H) 15 ABL1 (T315I) ABL1 (T315I) ABL1 (T315I) 35 ABL1 (Y253F) ABL1(Y253F) ABL1 (Y253F) 23 ABL2 ABL2 ABL2 43 ACVR1 ACVR1 ACVR1 61 ACVR1BACVR1B ACVR1B 63 ACVR2A ACVR2A ACVR2A 92 ACVR2B ACVR2B ACVR2B 63 ACVRL1ACVRL1 ACVRL1 91 ADCK3 ADCK3 ADCK3 14 ADCK4 ADCK4 ADCK4 14 AKT1 AKT1AKT1 100 AKT2 AKT2 AKT2 100 AKT3 AKT3 AKT3 66 ALK ALK ALK 10 AMPK-alpha1AMPK-alpha1 AMPK-alpha1 50 AMPK-alpha2 AMPK-alpha2 AMPK-alpha2 61 ANKK1ANKK1 ANKK1 73 ARK5 ARK5 ARK5 29 ASK1 ASK1 ASK1 100 AURKA AURKA AURKA100 AURKB AURKB AURKB 56 AURKC AURKC AURKC 47 AXL AXL AXL 25 BIKE BIKEBIKE 36 BLK BLK BLK 4.4 BMPR1A BMPR1A BMPR1A 100 BMPR1B BMPR1B BMPR1B 78BMPR2 BMPR2 BMPR2 100 BMX BMX BMX 73 BRAF BRAF BRAF 81 BRAF (V600E) BRAF(V600E) BRAF (V600E) 73 BRK BRK BRK 46 BRSK1 BRSK1 BRSK1 53 BRSK2 BRSK2BRSK2 33 BTK BTK BTK 77 CAMK1 CAMK1 CAMK1 65 CAMK1D CAMK1D CAMK1D 83CAMK1G CAMK1G CAMK1G 75 CAMK2A CAMK2A CAMK2A 63 CAMK2B CAMK2B CAMK2B 77CAMK2D CAMK2D CAMK2D 35 CAMK2G CAMK2G CAMK2G 58 CAMK4 CAMK4 CAMK4 92CAMKK1 CAMKK1 CAMKK1 71 CAMKK2 CAMKK2 CAMKK2 60 CDC2L1 CDC2L1 CDC2L1 42CDC2L2 CDC2L2 CDC2L2 37 CDK11 CDK11 CDK11 87 CDK2 CDK2 CDK2 94 CDK3 CDK3CDK3 91 CDK5 CDK5 CDK5 100 CDK7 CDK7 CDK7 0.55 CDK8 CDK8 CDK8 100 CDK9CDK9 CDK9 86 CDKL2 CDKL2 CDKL2 67 CHEK1 CHEK1 CHEK1 42 CHEK2 CHEK2 CHEK238 CIT CIT CIT 5.0 CLK1 CLK1 CLK1 31 CLK2 CLK2 CLK2 64 CLK3 CLK3 CLK3 45

CHART 1.2 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit GeneSymbol Clone ID (Primary) % Ctrl @ 10000 nM CLK4 CLK4 CLK4 17 CSF1RCSF1R CSF1R 50 CSK CSK CSK 53 CSNK1A1L CSNK1A1L CSNK1A1L 44 CSNK1DCSNK1D CSNK1D 25 CSNK1E CSNK1E CSNK1E 9.6 CSNK1G1 CSNK1G1 CSNK1G1 55CSNK1G2 CSNK1G2 CSNK1G2 76 CSNK1G3 CSNK1G3 CSNK1G3 61 CSNK2A1 CSNK2A1CSNK2A1 56 CSNK2A2 CSNK2A2 CSNK2A2 52 DAPK1 DAPK1 DAPK1 26 DAPK2 DAPK2DAPK2 61 DAPK3 DAPK3 DAPK3 34 DCAMKL1 DCAMKL1 DCAMKL1 92 DCAMKL2 DCAMKL2DCAMKL2 69 DCAMKL3 DCAMKL3 DCAMKL3 14 DDR1 DDR1 DDR1 39 DDR2 DDR2 DDR276 DLK DLK DLK 92 DMPK DMPK DMPK 6.8 DMPK2 DMPK2 DMPK2 61 DRAK1 DRAK1DRAK1 51 DRAK2 DRAK2 DRAK2 58 DYRK1B DYRK1B DYRK1B 66 EGFR EGFR EGFR 0EGFR (E746-A750del) EGFR (E746-A750del) EGFR (E746-A750del) 0 EGFR(G719C) EGFR (G719C) EGFR (G719C) 0 EGFR (G719S) EGFR (G719S) EGFR(G719S) 0 EGFR (L747-E749del, A750P) EGFR (L747-E749del, A750P) EGFR(L747-E749del, A750P) 0 EGFR (L747-S752del, P753S) EGFR (L747-S752del,P753S) EGFR (L747-S752del, P753S) 0.1 EGFR (L747-T751del, Sins) EGFR(L747-T751del, Sins) EGFR (L747-T751del, Sins) 0 EGFR (L858R) EGFR(L858R) EGFR (L858R) 0 EGFR (L861Q) EGFR (L861Q) EGFR (L861Q) 0 EGFR(S752-1759del) EGFR (S752-1759del) EGFR (S752-1759del) 0 EPHA1 EPHA1EPHA1 55 EPHA2 EPHA2 EPHA2 100 EPHA3 EPHA3 EPHA3 56 EPHA4 EPHA4 EPHA4 64EPHA5 EPHA5 EPHA5 86 EPHA6 EPHA6 EPHA6 25 EPHA7 EPHA7 EPHA7 70 EPHA8EPHA8 EPHA8 66 EPHB1 EPHB1 EPHB1 72 EPHB2 EPHB2 EPHB2 83 EPHB3 EPHB3EPHB3 75 EPHB4 EPHB4 EPHB4 39 ERBB2 ERBB2 ERBB2 31 ERBB4 ERBB4 ERBB4 3.3ERK1 ERK1 ERK1 98 ERK2 ERK2 ERK2 100 ERK3 ERK3 ERK3 10 ERK4 ERK4 ERK43.7 ERK5 ERK5 ERK5 90 ERK8 ERK8 ERK8 62 FAK FAK FAK 85 FER FER FER 94FES FES FES 72 FGFR1 FGFR1 FGFR1 64 FGFR2 FGFR2 FGFR2 100 FGFR3 FGFR3FGFR3 100 FGFR3 (G697C) FGFR3 (G697C) FGFR3 (G697C) 82 FGFR4 FGFR4 FGFR469 FGR FGR FGR 19 FLT1 FLT1 FLT1 64 FLT3 FLT3 FLT3 49 FLT3 (D835H) FLT3(D835H) FLT3 (D835H) 8.1 FLT3 (D835Y) FLT3 (D835Y) FLT3 (D835Y) 9.4 FLT3(ITD) FLT3 (ITD) FLT3 (ITD) 0.1

CHART 1.3 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit GeneSymbol Clone ID (Primary) % Ctrl@10000 nM FLT3 (K663Q) FLT3 (K663Q) FLT3(K663Q) 56 FLT3 (N8411) FLT3 (N8411) FLT3 (N8411) 16 FLT4 FLT4 FLT4 66FRK FRK FRK 10 FYN FYN FYN 41 GAK GAK GAK 0.95 GCN2 (Kin.Dom.2.S808G)GCN2 (Kin.Dom.2.S808G) GCN2 (Kin.Dom.2.S808G) 12 GSK3A GSK3A GSK3A 75GSK3B GSK3B GSK3B 100 HCK HCK HCK 17 HIPK1 HIPK1 HIPK1 70 HPK1 HPK1 HPK147 IGF1R IGF1R IGF1R 74 IKK-alpha IKK-alpha IKK-alpha 41 IKK-betaIKK-beta IKK-beta 51 IKK-epsilon IKK-epsilon IKK-epsilon 75 INSR INSRINSR 90 INSRR INSRR INSRR 79 IRAK3 IRAK3 IRAK3 25 ITK ITK ITK 82 JAK1(JH1domain-catalytic) JAK1 (JH1domain-catalytic) JAK1(JH1domain-catalytic) 15 JAK1 (JH2domain-pseudokinase) JAK1(JH2domain-pseudokinase) JAK1 (JH2domain-pseudokinase) 81 JAK2(JH1domain-catalytic) JAK2 (JH1domain-catalytic) JAK2(JH1domain-catalytic) 38 JAK3 (JH1domain-catalytic) JAK3(JH1domain-catalytic) JAK3 (JH1domain-catalytic) 72 JNK1 JNK1 JNK1 99JNK2 JNK2 JNK2 95 JNK3 JNK3 JNK3 77 KIT KIT KIT 26 KIT (D816V) KIT(D816V) KIT (D816V) 17 KIT (V559D) KIT (V559D) KIT (V559D) 17 KIT(V559D, T670I) KIT (V559D, T670I) KIT (V559D, T670I) 28 KIT (V559D,V654A) KIT (V559D, V654A) KIT (V559D, V654A) 47 LATS1 LATS1 LATS1 91LATS2 LATS2 LATS2 93 LCK LCK LCK 1.5 LIMK1 LIMK1 LIMK1 62 LIMK2 LIMK2LIMK2 100 LKB1 LKB1 LKB1 69 LOK LOK LOK 0 LTK LTK LTK 3.8 LYN LYN LYN 13MAP3K3 MAP3K3 MAP3K3 5.9 MAP3K4 MAP3K4 MAP3K4 53 MAP4K2 MAP4K2 MAP4K2 48MAP4K3 MAP4K3 MAP4K3 52 MAP4K4 MAP4K4 MAP4K4 20 MAP4K5 MAP4K5 MAP4K5 23MAPKAPK2 MAPKAPK2 MAPKAPK2 90 MAPKAPK5 MAPKAPK5 MAPKAPK5 79 MARK1 MARK1MARK1 56 MARK2 MARK2 MARK2 70 MARK3 MARK3 MARK3 62 MARK4 MARK4 MARK4 100MEK1 MEK1 MEK1 50 MEK2 MEK2 MEK2 44 MEK3 MEK3 MEK3 100 MEK4 MEK4 MEK4 92MEK6 MEK6 MEK6 75 MELK MELK MELK 57 MERTK MERTK MERTK 48 MET MET MET 33MINK MINK MINK 16 MKNK1 MKNK1 MKNK1 1.2 MKNK2 MKNK2 MKNK2 16 MLCK MLCKMLCK 77 MLK1 MLK1 MLK1 100 MLK2 MLK2 MLK2 83 MLK3 MLK3 MLK3 72 MRCKAMRCKA MRCKA 81

CHART 1.4 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit GeneSymbol Clone ID (Primary) % Ctrl @ 10000 nM MRCKB MRCKB MRCKB 71 MST1MST1 MST1 100 MST1R MST1R MST1R 94 MST2 MST2 MST2 85 MST3 MST3 MST3 42MST4 MST4 MST4 29 MUSK MUSK MUSK 100 MYLK MYLK MYLK 62 MYLK2 MYLK2 MYLK24.8 MYO3A MYO3A MYO3A 70 MYO3B MYO3B MYO3B 100 NDR2 NDR2 NDR2 74 NEK1NEK1 NEK1 66 NEK2 NEK2 NEK2 63 NEK5 NEK5 NEK5 94 NEK6 NEK6 NEK6 94 NEK7NEK7 NEK7 78 NEK9 NEK9 NEK9 85 NLK NLK NLK 55 p38-alpha p38-alphap38-alpha 89 p38-beta p38-beta p38-beta 100 p38-delta p38-deltap38-delta 100 p38-gamma p38-gamma p38-gamma 97 PAK1 PAK1 PAK1 74 PAK2PAK2 PAK2 67 PAK3 PAK3 PAK3 55 PAK4 PAK4 PAK4 80 PAK6 PAK6 PAK6 70 PAK7PAK7 PAK7 63 PCTK1 PCTK1 PCTK1 100 PCTK2 PCTK2 PCTK2 72 PCTK3 PCTK3PCTK3 82 PDGFRA PDGFRA PDGFRA 50 PDGFRB PDGFRB PDGFRB 10 PDPK1 PDPK1PDPK1 94 PFTAIRE2 PFTAIRE2 PFTAIRE2 71 PFTK1 PFTK1 PFTK1 71 PHKG1 PHKG1PHKG1 50 PHKG2 PHKG2 PHKG2 59 PIK3C2B PIK3C2B PIK3C2B 93 PIK3CA PIK3CAPIK3CA 100 PIK3CA (E545K) PIK3CA (E545K) PIK3CA (E545K) 100 PIK3CBPIK3CB PIK3CB 100 PIK3CD PIK3CD PIK3CD 89 PIK3CG PIK3CG PIK3CG 78 PIM1PIM1 PIM1 39 PIM2 PIM2 PIM2 83 PIM3 PIM3 PIM3 23 PIP5K1A PIP5K1A PIP5K1A61 PIP5K2B PIP5K2B PIP5K2B 49 PKAC-alpha PKAC-alpha PKAC-alpha 100PKAC-beta PKAC-beta PKAC-beta 88 PKMYT1 PKMYT1 PKMYT1 74 PKN1 PKN1 PKN150 PKN2 PKN2 PKN2 31 PLK1 PLK1 PLK1 81 PLK3 PLK3 PLK3 97 PLK4 PLK4 PLK470 PRKCD PRKCD PRKCD 84 PRKCE PRKCE PRKCE 65 PRKCH PRKCH PRKCH 83 PRKCQPRKCQ PRKCQ 83 PRKD1 PRKD1 PRKD1 9.6 PRKD2 PRKD2 PRKD2 0.4 PRKD3 PRKD3PRKD3 7.2 PRKG1 PRKG1 PRKG1 75 PRKG2 PRKG2 PRKG2 40 PRKR PRKR PRKR 51PRKX PRKX PRKX 100

CHART 1.5 Kinase Target NRC-2694 Descriptive Gene Symbol Ambit GeneSymbol Clone ID (Primary) % Ctrl @ 10000 nM PYK2 PYK2 PYK2 71 RAF1 RAF1RAF1 100 RET RET RET 65 RET (M918T) RET (M918T) RET (M918T) 37 RET(V804L) RET (V804L) RET (V804L) 81 RET (V804M) RET (V804M) RET (V804M)100 RIOK1 RIOK1 RIOK1 36 RIOK2 RIOK2 RIOK2 43 RIOK3 RIOK3 RIOK3 37 RIPK1RIPK1 RIPK1 100 RIPK2 RIPK2 RIPK2 2.4 RIPK4 RIPK4 RIPK4 96 ROCK2 ROCK2ROCK2 38 ROS1 ROS1 ROS1 46 RPS6KA1 (Kin.Dom.-1-N-terminal) RPS6KA1(Kin.Dom.-1-N-terminal) RPS6KA1 (Kin.Dom.-1-N-terminal) 100 RPS6KA1(Kin.Dom.-2-C-terminal) RPS6KA1 (Kin.Dom.-2-C-terminal) RPS6KA1(Kin.Dom.-2-C-terminal) 49 RPS6KA2 (Kin.Dom.-1-N-terminal) RPS6KA2(Kin.Dom.-1-N-terminal) RPS6KA2 (Kin.Dom.-1-N-terminal) 70 RPS6KA2(Kin.Dom.-2-C-terminal) RPS6KA2 (Kin.Dom.-2-C-terminal) RPS6KA2(Kin.Dom.-2-C-terminal) 78 RPS6KA3 (Kin.Dom.-1-N-terminal) RPS6KA3(Kin.Dom.-1-N-terminal) RPS6KA3 (Kin.Dom.-1-N-terminal) 94 RPS6KA4(Kin.Dom.-1-N-terminal) RPS6KA4 (Kin.Dom.-1-N-terminal) RPS6KA4(Kin.Dom.-1-N-terminal) 84 RPS6KA4 (Kin.Dom.-2-C-terminal) RPS6KA4(Kin.Dom.-2-C-terminal) RPS6KA4 (Kin.Dom.-2-C-terminal) 8.9 RPS6KA5(Kin.Dom.-1-N-terminal) RPS6KA5 (Kin.Dom.-1-N-terminal) RPS6KA5(Kin.Dom.-1-N-terminal) 85 RPS6KA5 (Kin.Dom.-2-C-terminal) RPS6KA5(Kin.Dom.-2-C-terminal) RPS6KA5 (Kin.Dom.-2-C-terminal) 57 RPS6KA6(Kin.Dom.-1-N-terminal) RPS6KA6 (Kin.Dom.-1-N-terminal) RPS6KA6(Kin.Dom.-1-N-terminal) 94 RPS6KA6 (Kin.Dom.-2-C-terminal) RPS6KA6(Kin.Dom.-2-C-terminal) RPS6KA6 (Kin.Dom.-2-C-terminal) 55 SgK085 SgK085SgK085 84 SgK110 SgK110 SgK110 83 SIK SIK SIK 53 SIK2 SIK2 SIK2 21 SLKSLK SLK 0.3 SNARK SNARK SNARK 19 SRC SRC SRC 24 SRMS SRMS SRMS 100 SRPK1SRPK1 SRPK1 40 SRPK2 SRPK2 SRPK2 73 SRPK3 SRPK3 SRPK3 38 STK16 STK16STK16 100 STK33 STK33 STK33 43 STK35 STK35 STK35 52 STK36 STK36 STK364.6 SYK SYK SYK 56 TAK1 TAK1 TAK1 66 TAOK1 TAOK1 TAOK1 54 TAOK3 TAOK3TAOK3 76 TEC TEC TEC 90 TESK1 TESK1 TESK1 83 TGFBR1 TGFBR1 TGFBR1 83TGFBR2 TGFBR2 TGFBR2 86 TIE1 TIE1 TIE1 52 TIE2 TIE2 TIE2 76 TLK1 TLK1TLK1 77 TLK2 TLK2 TLK2 82 TNIK TNIK TNIK 17 TNK1 TNK1 TNK1 41 TNK2 TNK2TNK2 37 TNNI3K TNNI3K TNNI3K 7.6 TRKA TRKA TRKA 100 TRKB TRKB TRKB 73TRKC TRKC TRKC 80 TSSK1B TSSK1B TSSK1B 93 TTK TTK TTK 48 TXK TXK TXK 39TYK2 (JH1domain-catalytic) TYK2 (JH1domain-catalytic) TYK2(JH1domain-catalytic) 97 TYK2 (JH2domain-pseudokinase) TYK2(JH2domain-pseudokinase) TYK2 (JH2domain-pseudokinase) 23 TYRO3 TYRO3TYRO3 42 ULK1 ULK1 ULK1 100 ULK2 ULK2 ULK2 39 ULK3 ULK3 ULK3 62 VEGFR2VEGFR2 VEGFR2 95

CHART 1.6 Kinase Target Descriptive Clone ID NRC-2694 Gene Symbol AmbitGene Symbol (Primary) % Ctrl @ 10000 nM WEE1 WEE1 WEE1 74 WEE2 WEE2 WEE253 YANK2 YANK2 YANK2 68 YANK3 YANK3 YANK3 100 YES YES YES 41 YSK1 YSK1YSK1 69 ZAK ZAK ZAK 74 ZAP70 ZAP70 ZAP70 100

We claim:
 1. A method of treating drug resistant non-small cell lungcancer in a subject in need of treatment for drug resistant non-smallcell lung cancer, the method comprising: administering to the subject aneffective amount of a compound of formula (IA)

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the pharmaceutically acceptable salt is a monohydrochloride, adihydrochloride, or a mixture thereof.